This invention was made with government support under grant R01-DK36425 and R01-AR38454 awarded by the National Institutes of Health. The U.S. government may have certain rights in this invention.
Basal laminae (basement membranes) are sheet-like, cell-associated extracellular matrices that play a central role in cell growth, tissue development, and tissue maintenance. They are present in virtually all tissues, and appear in the earliest stages of embryonic development.
Basal laminae are central to a variety of architectural and cell-interactive functions (See for example, Malinda and Kleinman, Int. J. Biochem. Cell Biol. 28:957-959 (1996); Aumailley and Krieg, J. Invest. Dermatology 106:209-214 (1996)). For example:
1. They serve as architectural supports for tissues, providing adhesive substrata for cells.
2. They create perm-selective barriers between tissue compartments that impede the migration of cells and passively regulate the exchange of macromolecules. These properties are illustrated by the kidney glomerular basement membrane, which functions as an important filtration structure, creating an effective blood-tissue barrier that is not permeable to most proteins and cells.
3. Basal laminae create highly interactive surfaces that can promote cell migration and cell elongation during embryogenesis and wound repair. Following an injury, they provide a surface upon which cells regenerate to restore normal tissue function.
4. Basal laminae present information encoded in their structure to contacting cells that is important for differentiation and tissue maintenance. This information is communicated to the cells through various receptors that include the integrins, dystroglycan, and cell surface proteoglycans. Signaling is dependent not only on the presence of matrix ligands and corresponding receptors that interact with sufficient affinities, but also on such topographical factors as ligand density in a three-dimensional matrix xe2x80x9clandscapexe2x80x9d, and on the ability of basal lamina components to cluster receptors. Because these matrix proteins can be long-lived, basal laminae create a xe2x80x9csurface memoryxe2x80x9d in the basal lamina for resident and transient cells.
The basal lamina is largely composed of laminin and type IV collagen heterotrimers that in turn become organized into complex polymeric structures. To date, six type IV collagen chains and at least twelve laminin subunits have been identified. These chains possess shared and unique functions and are expressed with specific temporal (developmental) and spatial (tissue-site specific) patterns.
Laminins are a family of heterotrimeric glycoproteins that reside primarily in the basal lamina. They function via binding interactions with neighboring cell receptors, and by forming laminin networks, and they are important signaling molecules that can strongly influence cellular function. Laminins are important in both maintaining cell/tissue phenotype as well as promoting cell growth and differentiation in tissue repair and development.
Laminins are large, multi-domain proteins, with a common structural organization. The laminin molecule integrates various matrix and cell interactive functions into one molecule.
Laminin molecules are comprised of an xcex1-, xcex2-, and xcex3-chain subunit joined together through a coiled-coil domain. Within this structure are identifiable domains that possess binding activity towards other laminin and basal lamina molecules, and membrane-bound receptors. Domains VI, IVb, and IVa form globular structures, and domains V, IIIb, and IIIa (which contain cysteine-rich EGF-like elements) form rod-like structures. (Kamiguchi et al., Ann. Rev. Neurosci. 21:97-125 (1998)) Domains I and II of the three chains participate in the formation of a triple-stranded coiled-coil structure (the long arm).
Table 1 shows the individual chains that each laminin type is composed of:
Four structurally-defined family groups of laminins have been identified. The first group of five identified laminin molecules all share the xcex21 and xcex31 chains, and vary by their xcex1-chain composition (xcex11 to xcex15 chain). The second group of five identified laminin molecules all share the xcex22 and xcex31 chain, and again vary by their xcex1-chain composition. The third group of identified laminin molecules has one identified member, laminin 5, with a chain composition of xcex13xcex23xcex32. The fourth group of identified laminin molecules has one identified member, laminin 12, with the newly identified xcex33 chain (xcex12xcex21xcex33)
Some progress has been made in elucidating the relationship between domain structure and function. (See, for example, Wewer and Engvall, Neuromusc. Disord. 6:409-418 (1996).) The overall sequence similarity among the homologous domains in different chains varies, but it is highest in domain VI (thought to play a key role in laminin polymerization), followed by domains V (possibly involved in protein-protein interactions) and III (entactin/nidogen binding; possible cell adhesion sites), and is lowest in domains I, II (both thought to be involved in intermolecular assembly, and containing possible cell adhesion sites), and G. Not all domains are present in all 3 types of chains. The globular G domain (thought to be involved in cell receptor binding) is present only in the xcex1 chains. Other domains may not be present in all chains within a certain chain type. For example, domain VI is absent from xcex13, xcex14, and xcex32 chains. (Wewer and Engvall, 1996)
As a result of their large size ( greater than 600 kD) and unique structure, laminin molecules can be resolved in the electron microscope. (Wewer and Engvall, 1996) Typically, laminins appear as cross-shaped molecules in an electron micrograph. The three short arms of the cross represent the amino terminal portions of each of the three separate laminin chains (one short arm per chain). The long arm of the cross is composed of the C-terminal parts of the three chains, which together form a coiled coil structure. (Wewer and Engvall, 1996) The long arm ends with the globular G domain.
The coiled-coil domain of the long arm is crucial for assembly of the three chains of laminin. (Yurchenco et al., Proc. Natl. Acad. Sci. 94:10189-10194 (1997)). Disulfide bonds bridge and stabilize all three chains in the most proximal region of the long arm and join the xcex2 and xcex3 chains in the most distal region of the long arm.
A model of laminin receptor-facilitated self-assembly, based on studies conducted with cultured skeletal myotubes and Schwann cells, predicts that laminins bind to their receptors, which freely diffuse in a fluidic membrane when ligand-free. Receptor engagement forces the laminins into a high local two-dimensional concentration, facilitating their mass-action driven assembly into ordered surface polymers. In this process, the engaged receptors are also reorganized, accompanied by cytoskeletal rearrangements. (Colognato, J. Cell Biol. 145:619-631 (1999)) This reorganization activates the receptors, causing signal transduction with the alteration of cell expression, shape and/or behavior.
One class of laminin receptors are the integrins, which are cell surface receptors that mediate many cell-matrix and cell-cell interactions. Integrins are heterodimers, consisting of an xcex1 and a xcex2 subunit. 16xcex1- and 8xcex2-subunits are known, and at least 22 combinations of xcex1 and xcex2 subunits have been identified to date. Some integrins have only one or a few known ligands, whereas others appear to be very promiscuous. Binding to integrins is generally of low affinity, and is dependent on divalent cations. Integrins, activated through binding to their ligands, transduce signals via kinase activation cascades, such as focal adhesion and mitogen-activated kinases. Several different integrins bind different laminin isoforms more or less specifically. (Aumailley et al., In The Laminins, Timpl and Ekblom, eds., Harwood Academic Publishers, Amsterdam. pp. 127-158 (1996))
Laminin 2 is composed of xcex22 (400 kD), xcex21 (approximately 100 kD), and xcex31 (approximately 100 kD) chains. The C-terminal G domain of the xcex22 chain forms a large globular structure responsible for binding to xcex1-dystroglycan. (Kamiguchi et al., 1998).
The short arm domains of laminin 1 are involved in the self-aggregation process (Schittney and Yurchenco, J. Cell Biol. 110:825-832 (1990)) and with extracellular matrix components, such as type IV collagen. Homology between the xcex11 (laminin 1) and xcex12 chains is 58.6%. The significant homology between the xcex11 and xcex12 chains, especially in the N-terminal domains, and their identical xcex2 and xcex3 chains, suggest that laminin 2 has a similar structural organization to laminin 1. (Kamiguchi et al., 1998)
Laminin 2 was originally found in the basement membranes of the placenta, striated muscle, and Schwann cells. (Leivo and Engvall, Proc. Natl. Acad. Sci. USA 85:1544-1548 (1998)) In normal adults, laminin 2 is predominant in the basal lamina of skeletal muscle, where it serves to provide mechanical reinforcement to the sarcolemma by linking the extracellular matrix and the subsarcolemmal cytoskeleton. (Sanes et al., J. Cell Biol. 111:1685-1699 (1990)).
Genetic defects affecting the structure or expression of laminin 2 are the causes of a major type of congenital muscular dystrophy (CMD). Laminin 2 has been shown to be specifically required for stabilizing myotubes during skeletal muscle development, and for preventing apoptosis, which is believed to explain some of the pathological events observed in CMD. (Kamiguchi et al., 1998)
In vitro studies have demonstrated that partially purified laminin 2 is important for myotube survival and maintenance of phenotype. (Vachon et al., J. Cell Biol. 134:1483-1497 (1996)). In vivo experiments have shown partial laminin xcex12 chain restoration in a laminin xcex12 deficient, CMD animal model by primary muscle cell transplantation. (Vilquin et al., J. Cell Biol. 133:185-197)
Laminin 2 is also the predominant laminin isoform present in the endoneurial basement membrane of developing and mature peripheral nerves, and was shown to promote Schwann cell migration, neurite outgrowth, and neurite regeneration (Kamiguchi et al., 1998), as well as myelin formation by oligodendrocytes (Buttery et al., Mol. Cell. Neurosci. 14:199-212 (1999). The results of various experiments have indicated that laminin 2, rather than laminin 1, is important in Schwann cell/basal lamina interactions, especially at early developmental stages. (Kamiguchi et al., 1998) Other studies have demonstrated that partially purified laminin 2 promotes neuronal cell migration and axon outgrowth (Agius and Cochard, J. Neurosci. 18:328-338 (1998); Kamiguchi et al, 1998; U.S. Pat. Nos. 5,444,158; 5,872,231; 5,624,905; and 5,863,743; Bates and Meyer, Develop. Biol. 181:91-101 (1997)). In a laminin 2 deficient CMD animal model, CMD was accompanied by dysmyelination of peripheral motor nerves, indicating that laminin 2 plays an important role in peripheral myelinogenesis.
Partially purified laminin 2 has also been shown to promote cell migration and attachment to a substrate of a variety of cell types, particularly muscle cells and cells of neuronal origin. (U.S. Pat. No. 5,444,158; White et al., Am. J. Resp. Biol. 20:787-796 (1999); Engvall et al., Exp. Cell Res. 198:115-123 (1992))
It has also been demonstrated that the molecular basis of the neural tropism of Mycobacterium leprae is attributable to the specific binding of M. leprae to the G domain of the laminin xcex12 chain on Schwann cell-axon units, while xcex1-dystroglycan (xcex1DG) was shown to serve as a Schwann cell receptor for M. leprae. (Rambukkana et al., Science 282:2076-2079 (1998); Rambukkana et al., Cell 88:811-821 (1997)). Native xcex1DG was shown to competitively inhibit the laminin-2 mediated M. leprae binding to primary Schwann cells. (Rambukkana et al. 1998)
Thus, research and therapeutic applications for laminin 2 and fragments thereof include, but are not limited to, peripheral nervous system (PNS) nerve regeneration, treatment of degenerative muscle disorders, regulating angiogenesis, promoting cell attachment and migration, ex vivo cell therapy, improving the biocompatibility of medical devices, improving the xe2x80x9ctakexe2x80x9d of grafts, and preparing improved cell culture devices and media.
At present, there is not a means to isolate adequate substantially purified laminin 2 from cell or tissue sources for research or therapeutic purposes, nor has a means been developed for production of recombinant heterotrimeric laminin 2. Laminin 2 can be partially purified from either placenta, or, in lesser amounts, from skeletal muscle. Human placenta has provided the only source for obtaining up to several milligrams of protein. (Cheng et al., J. Biol. Chem. 272:31525-32, 1997) However preparations of this laminin normally contain about an equal molar quantity of laminin 4 (xcex12xcex22xcex31) and the protein nidogen (entactin). The nidogen is bound to the laminin through a fairly strong but non-covalent association. It is difficult to remove most of the laminin 4, and even after additional steps, a significant contaminating level of laminin 4 remains. Denaturing conditions are required to remove the nidogen.
Therefore, there is a need in the art for adequate amounts of substantially purified laminin-2, and methods for making laminin 2. A preferred method of production is the use of recombinant DNA technology to engineer a cell line of choice to produce recombinant laminin-2. A recombinant-based method of laminin-2 production has several advantages over purification from tissue or isolation from cell lines in culture:
1. The recombinantly produced protein is free of pathogens. While this is also true for endogenous cell culture produced protein, protein derived from human tissue carries a risk for contamination by HIV, hepatitis, and other infectious agents.
2. Expression levels of the protein, and hence yields, can be improved through the use of genetically engineered genes/vectors that enhance the production of the encoded protein.
3. It is possible to engineer additional peptide sequences to the protein chain that provides a binding site for a commercially viable affinity purification procedure. 4. The method can provide for the modification of protein structure/function through the addition, substitution, elimination, and/or other modifications of protein domain structures. For example, it may be desirable to introduce an integrin binding site (e.g. RGD), switch integrin recognition sites, or engineer in a stable binding site to a synthetic substrate. Thus, the creation of expression vectors that express laminin chains generates enormous flexibility for future uses and creates a basis for creating second generation xe2x80x9cdesignerxe2x80x9d laminins.
The present invention fulfills the need in the art for substantially purified laminin 2 protein, methods for making substantially purified recombinant laminin 2 (hereinafter referred to as r-laminin 2), and methods of using substantially purified laminin 2 for research and therapeutic purposes including, but not limited to, peripheral nerve regeneration, treatment of degenerative muscle disorders, angiogenesis regulation, promoting cell attachment and migration, ex vivo cell therapy, improving the xe2x80x9ctakexe2x80x9d of grafts, improving the biocompatibility of medical devices, and preparing improved cell culture devices and media
In one aspect, the present invention provides mammalian cells that have been transfected with expression vector(s) encoding at least one of the laminin xcex12, xcex21 and xcex31 chains, wherein the cells secrete r-laminin 2.
In another aspect, the present invention provides substantially purified laminin 2 and methods for producing r-laminin 2.
In a further embodiment, the present invention provides a novel, isolated laminin 2 xcex12 nucleic acid and xcex12 protein. In this embodiment, the protein product contains an additional 30 amino acids at its carboxyl terminus relative to the previously reported sequence.
In a further aspect, the present invention provides pharmaceutical compositions, comprising substantially purified laminin 2, or the novel recombinant xcex12 protein together with a pharmaceutically acceptable carrier. Such pharmaceutical compositions can optionally be provided with other compounds, such as extracellular matrix components.
The present invention further provides methods for peripheral nerve regeneration, treatment of degenerative muscle disorders, regulating angiogenesis, promoting cell attachment and migration, ex vivo cell therapy, improving the biocompatibility of medical devices, improving the xe2x80x9ctakexe2x80x9d of grafts, and preparing improved cell culture devices and media, comprising providing an amount effective of the substantially purified laminin 2, or pharmaceutical compositions thereof, for the desired outcome.
In a further aspect, the present invention provides improved medical devices or grafts, wherein the improvement comprises applying to the devices or grafts an amount effective of substantially purified laminin 2 or pharmaceutical compositions thereof, for the desired application. Such devices can optionally be provided with other compounds, such as extracellular matrix components to further improve the biocompatibility or the effectiveness of the medical device or graft.
In a further aspect, the invention provides improved cell culture devices, by providing an amount effective of substantially purified laminin 2, or pharmaceutical compositions thereof, for the attachment of cells to a cell culture device for the subsequent proliferation/differentiation/stasis of the cells.
In another aspect, the invention provides a cell culture growth supplement, comprising substantially purified laminin 2. In another aspect, the invention provides an improved cell culture growth media, wherein the improvement comprises the addition of substantially purified laminin 2 to the growth medium.